Lectin affinity chromatography of proteins bearing O-linked oligosaccharides: application of jacalin-agarose

Anal Biochem. 1990 Aug 1;188(2):271-7. doi: 10.1016/0003-2697(90)90605-9.

Abstract

The lectin jacalin immobilized on agarose was found to bind a variety of glycoproteins known to contain typical O-linked oligosaccharides, including human IgA, C1 inhibitor, chorionic gonadotropin, plasminogen, bovine protein Z, bovine coagulation factor X, and fetuin. These proteins were eluted from columns of jacalin-agarose specifically by alpha-galactopyranosides such as melibiose and alpha-methylgalactopyranoside but not by lactose or other sugars. Treatment of asialofetuin with endo--alpha--N--acetylgalactosaminidase eliminated its affinity for the lectin column, and other proteins known to contain only N-linked oligosaccharides such as ovalbumin, transferrin, and alpha 1-acid glycoprotein were not retained by the lectin. Binding of proteins with O-linked oligosaccharides to the lectin column did not require divalent cations and was affected little by changes in pH and ionic strength over a wide range. Virtually all of the glycosidically linked oligosaccharides of fetuin, chorionic gonadotropin, and plasminogen are known to be sialated. Thus, binding of these glycoproteins to jacalin, which is known to have affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides of these proteins, was not prevented by the presence of sialic acids. Affinity of oligosaccharides for jacalin did appear to be reduced by occurrence of sialic acids as it was found that higher concentrations of melibiose were required to elute asialofetuin than fetuin from jacalin-agarose. Results of the present study indicate that affinity chromatography using this lectin is a widely applicable technique for identifying and purifying proteins bearing O-linked oligosaccharides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Proteins / analysis
  • Blood Proteins / isolation & purification
  • Carbohydrate Sequence
  • Cattle
  • Chorionic Gonadotropin / analysis
  • Chorionic Gonadotropin / isolation & purification
  • Chromatography, Affinity / methods*
  • Factor X / analysis
  • Factor X / isolation & purification
  • Glycoproteins / analysis
  • Glycoproteins / isolation & purification*
  • Humans
  • Hydrogen-Ion Concentration
  • Immunoglobulin A / analysis
  • Immunoglobulin A / isolation & purification
  • Lectins*
  • Molecular Sequence Data
  • Oligosaccharides / analysis*
  • Plant Lectins*
  • Plasminogen Inactivators / analysis
  • Plasminogen Inactivators / isolation & purification
  • Sialic Acids
  • alpha-Fetoproteins / analysis
  • alpha-Fetoproteins / isolation & purification

Substances

  • Blood Proteins
  • Chorionic Gonadotropin
  • Glycoproteins
  • Immunoglobulin A
  • Lectins
  • Oligosaccharides
  • Plant Lectins
  • Plasminogen Inactivators
  • Sialic Acids
  • alpha-Fetoproteins
  • jacalin
  • plasma protein Z
  • Factor X